Antibiotic-resistant Gram-positive bacteria, such as Enterococcus faecalis and Staphylococcus aureus, are major causes of hospital-acquired infections. E. faecalis is a successful hospital-acquired pathogen in part because of its resistance to commonly used antibiotics that target the bacterial cell envelope. However, the genetic and biochemical basis for this antimicrobial resistance in E. faecalis is poorly understood. Preliminary studies identified numerous genes involved in nucleotide metabolism that influence antimicrobial resistance in E. faecalis. These observations suggest that intracellular nucleotide levels are playing critical, as-yet-unappreciated roles in regulation of cellular processes leading to antibiotic resistance. Our long-term goal is to understand the biological and biochemical roles of intracellular nucleotides in antibiotic resistance, and more generally, the overall integrated genetic and biochemical basis for antimicrobial resistance in E. faecalis. The objective of this proposal is to define which specific nucleotides influence resistance, and to begin elucidating how nucleotides exert their effect on resistance by identifying nucleotide-responsive factors encoded in the E. faecalis genome. To achieve this goal, we will: 1) Define the relationship of nucleotide levels to cephalosporin resistance in E. faecalis by measuring nucleotide levels in mutants exhibiting altered cephalosporin resistance; and 2) Exploit unbiased, genome-wide genetic strategies to identify nucleotide-responsive effectors that influence cephalosporin resistance. By achieving these aims, the research proposed here will provide insights into the role of nucleotides in regulation of cellular processe in E. faecalis, enhance fundamental understanding of mechanisms responsible for antimicrobial resistance in Gram- positive bacteria, and form the foundation for future efforts to develop innovative therapies against infections caused by resistant bacteria.